Molecular investigation of feline coronavirus (FCoV) in local pet cats

Feline coronavirus (FCoV) infection is a very common in cat population. FCoV is further classified into two biotypes namely feline enteric coronavirus (FECV) and mutated feline infectious peritonitis virus (FIPV), in which FIPV causes a fatal immune complex disease by changing the tropism from enterocytes to monocytes. Previous studies on molecular detection of FCoV in cats were carried out in catteries but limited study investigate the presence of FCoV antigen in local pet cats. By considering this fact, this study aims to detect FCoV antigen via RT-PCR assay in local pet cats and to compare the similarity of the identified FCoV strain with previous related virus by phylogenetic analysis. By using convenience sampling, rectal swabs and buffy coat were collected from 16 clinically ill pet cats and 5 healthy pet cats. Viral RNA was extracted and subjected to one-step RT-PCR, targeting polymerase gene. Only one out of 21 fecal samples was positive for FCoV and none from buffy coat samples. Phylogenetic analysis revealed that the identified positive sample was highly homologous, up to 95%, to FCoV strain from Netherlands and South Korea on partial sequence of polymerase gene. In conclusion, this study detected FCoV antigen in local pet cats from fecal samples while negative detection from fecal and buffy coat samples could not completely rule out the possibilities of FCoV infection due to the complexity of the virus diagnosis that require multiple series of analysis

Immunological analysis of Nodavirus capsid displaying the domain III of Japanese Encephalitis Virus evelope protein

Japanese encephalitis virus (JEV) is the pathogen that causes Japanese encephalitis (JE) in humans and horses. Lethality of the virus was reported to be between 20–30%, of which, 30–50% of the JE survivors develop neurological and psychiatric sequelae. Attributed to the low effectiveness of current therapeutic approaches against JEV, vaccination remains the only effective approach to prevent the viral infection. Currently, live-attenuated and chimeric-live vaccines are widely used worldwide but these vaccines pose a risk of virulence restoration. Therefore, continuing development of JE vaccines with higher safety profiles and better protective efficacies is urgently needed. In this study, the Macrobrachium rosenbergii nodavirus (MrNV) capsid protein (CP) fused with the domain III of JEV envelope protein (JEV-DIII) was produced in Escherichia coli. The fusion protein (MrNV-CPJEV-DIII) assembled into virus-like particles (VLPs) with a diameter of approximately 18 nm. The BALB/c mice injected with the VLPs alone or in the presence of alum successfully elicited the production of anti-JEV-DIII antibody, with titers significantly higher than that in mice immunized with IMOJEV, a commercially available vaccine. Immunophenotyping showed that the MrNV-CPJEV-DIII supplemented with alum triggered proliferation of cytotoxic T-lymphocytes, macrophages, and natural killer (NK) cells. Additionally, cytokine profiles of the immunized mice revealed activities of cytotoxic T-lymphocytes, macrophages, and NK cells, indicating the activation of adaptive cellular and innate immune responses mediated by MrNV-CPJEV-DIII VLPs. Induction of innate, humoral, and cellular immune responses by the MrNV-CPJEV-DIII VLPs suggest that the chimeric protein is a promising JEV vaccine candidate

Prevalence and risk factors of Japanese encephalitis virus (JEV) in livestock and companion animal in high-risk areas in Malaysia

Japanese encephalitis (JE) is vector-borne zoonotic disease which causes encephalitis in humans and horses. Clinical signs for Japanese encephalitis virus (JEV) infection are not clearly evident in the majority of affected animals. In Malaysia, information on the prevalence of JEV infection has not been established. Thus, a cross-sectional study was conducted during two periods, December 2015 to January 2016 and March to August in 2016, to determine the prevalence and risk factors in JEV infections among animals and birds in Peninsular Malaysia. Serum samples were harvested from the 416 samples which were collected from the dogs, cats, water birds, village chicken, jungle fowls, long-tailed macaques, domestic pigs, and cattle in the states of Selangor, Perak, Perlis, Kelantan, and Pahang. The serum samples were screened for JEV antibodies by commercial IgG ELISA kits. A questionnaire was also distributed to obtain information on the animals, birds, and the environmental factors of sampling areas. The results showed that dogs had the highest seropositive rate of 80% (95% CI: ± 11.69) followed by pigs at 44.4% (95% CI: ± 1.715), cattle at 32.2% (95% CI: ± 1.058), birds at 28.9% (95% CI: ± 5.757), cats at 15.6% (95% CI: ± 7.38), and monkeys at 14.3% (95% CI: ± 1.882). The study also showed that JEV seropositivity was high in young animals and in areas where mosquito vectors and migrating birds were prevalent

The distribution of important sero-complexes of flaviviruses in Malaysia

Flaviviruses (FVs) are arthropod-borne viruses of medical and veterinary importance. Numerous species of FVs have been isolated from various host; mainly humans, animals, ticks, and mosquitoes. Certain FVs are extremely host-specific; at the same time, some FVs can infect an extensive range of species. Based on published literatures, 11 species of FVs have been detected from diverse host species in Malaysia. In humans, dengue virus and Japanese encephalitis virus have been reported since 1901 and 1942. In animals, the Batu Cave virus, Sitiawan virus, Carey Island, Tembusu virus, Duck Tembusu virus, and Japanese encephalitis viruses were isolated from various species. In mosquitoes, Japanese encephalitis virus and Kunjin virus were isolated from Culex spp., while Zika virus and Jugra virus were isolated from Aedes spp. In ticks, the Langat virus was isolated from Ixodes spp. One of the major challenges in the diagnosis of FVs is the presence of sero-complexes as a result of cross-reactivity with one or more FV species. Subsequently, the distribution of specific FVs among humans and animals in a specific population is problematic to assess and often require comprehensive and thorough analyses. Molecular assays such as quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) and digital droplet RT-PCR (ddRT-PCR) have been used for the differentiation of flavivirus infections to increase the accuracy of epidemiological data for disease surveillance, monitoring, and control. In situations where sero-complexes are common in FVs, even sensitive assays such as qRT-pCR can produce false positive results. In this write up, an overview of the various FV sero-complexes reported in Malaysia to date and the challenges faced in diagnosis of FV infections are presented

Japanese Encephalitis in Malaysia: an overview and timeline

Japanese encephalitis (JE) is a vector-borne zoonotic disease caused by the Japanese encephalitis virus (JEV). It causes encephalitis in human and horses, and may lead to reproductive failure in sows. The first human encephalitis case in Malaya (now Malaysia) was reported during World War II in a British prison in 1942. Later, encephalitis was observed among race horses in Singapore. In 1951, the first JEV was isolated from the brain of an encephalitis patient. The true storyline of JE exposure among humans and animals has not been documented in Malaysia. In some places such as Sarawak, JEV has been isolated from mosquitoes before an outbreak in 1992. JE is an epidemic in Malaysia except Sarawak. There are four major outbreaks reported in Pulau Langkawi (1974), Penang (1988), Perak and Negeri Sembilan (1998–1999), and Sarawak (1992). JE is considered endemic only in Sarawak. Initially, both adults and children were victims of JE in Malaysia, however, according to the current reports; JE infection is only lethal to children in Malaysia. This paper describes a timeline of JE cases (background of each case) from first detection to current status, vaccination programs against JE, diagnostic methods used in hospitals and factors which may contribute to the transmission of JE among humans and animals in Malaysia

Prevalence and risk factors of Japanese encephalitis virus (JEV) in livestock and companion animal in high-risk areas in Malaysia

Japanese encephalitis (JE) is vector-borne zoonotic disease which causes encephalitis in humans and horses. Clinical signs for Japanese encephalitis virus (JEV) infection are not clearly evident in the majority of affected animals. In Malaysia, information on the prevalence of JEV infection has not been established. Thus, a cross-sectional study was conducted during two periods, December 2015 to January 2016 and March to August in 2016, to determine the prevalence and risk factors in JEV infections among animals and birds in Peninsular Malaysia. Serum samples were harvested from the 416 samples which were collected from the dogs, cats, water birds, village chicken, jungle fowls, long-tailed macaques, domestic pigs, and cattle in the states of Selangor, Perak, Perlis, Kelantan, and Pahang. The serum samples were screened for JEV antibodies by commercial IgG ELISA kits. A questionnaire was also distributed to obtain information on the animals, birds, and the environmental factors of sampling areas. The results showed that dogs had the highest seropositive rate of 80% (95% CI: ± 11.69) followed by pigs at 44.4% (95% CI: ± 1.715), cattle at 32.2% (95% CI: ± 1.058), birds at 28.9% (95% CI: ± 5.757), cats at 15.6% (95% CI: ± 7.38), and monkeys at 14.3% (95% CI: ± 1.882). The study also showed that JEV seropositivity was high in young animals and in areas where mosquito vectors and migrating birds were prevalent

Detection of Japanese Encephalitis virus in free roamer cats in Malaysia

lntroduction: Japanese Encephalitis virus (JEV) is transmitted by mosquitoes of lhe Culex species and responsible of viral encephalitis in Asia and it is endemic in Malaysia since 1998. Cats and dogs are reported to be susceptible to JEV infection but high positivity was reported in the latter species. The study is conducted to determine if local cats are susceptible to infection by circulating JEV. Methods: The free roaming cats in Klang Valley were chosen since they are possibly exposed to the mosquitoes throughout their life span as compared to indoor cats. About 3ml blood were withdrawn from L2 Domestic Short Hair cats through jugular venipuncture (IACUC No. UPM/IACUC/AUP-R008/2015). Blood plasma was subjected to two steps RT-PCR assay by using non-structural protein NS3 region, and sequenced. Result: Of 12 blood samples, five were positive for JEV with PCR product of approximately 600 bps. Phylogenetic analyses of three amplicons revealed that the local strains are clade together with 97% bootstrap value with JEV strain Nakayama (HE861351); iEV isolated from Rousettus leschenaulti (JF7052S5 and FJ185036) and from Murino ouroto (FJL85037) in which both are reported in China. Conclusion: Free roamer cats are susceptible to JEV infection. More cats should be screened for iEV to determine their seropositivity. Serosurvey of companion animals may accurately reflect risk of humans since these animals are normally owned by household and cats may be one of the sentinel animals for surveillance

Evidence of West Nile virus infection in migratory and resident wild birds in west coast of peninsular Malaysia

West Nile virus (WNV) is a zoonotic mosquito-borne flavivirus that is harbored and amplified by wild birds via the enzootic transmission cycle. Wide range of hosts are found to be susceptible to WNV infection including mammals, amphibians and reptiles across the world. Several studies have demonstrated that WNV was present in the Malaysian Orang Asli and captive birds. However, no data are available on the WNV prevalence in wild birds found in Malaysia. Therefore this study was conducted to determine the serological and molecular prevalence of WNV in wild birds in selected areas in the West Coast of Peninsular Malaysia. Two types of wild birds were screened, namely migratory and resident birds in order to explore any possibility of WNV transmission from the migratory birds to the resident birds. Thus, a cross-sectional study was conducted at the migratory birds sanctuary located in Kuala Gula, Perak and Kapar, Selangor by catching 163 migratory birds, and 97 resident birds from Kuala Gula and Parit Buntar, Perak at different time between 2016 and 2017 (Total, n = 260). Blood and oropharyngeal swabs were collected for serological and molecular analysis, respectively. Serum were screened for WNV antibodies using a commercial competitive ELISA (c-ELISA) (ID Screen® West Nile Competition Multi-species ELISA, ID VET, Montpellier, France) and cross-reactivity towards Japanese Encephalitis virus (JEV) was also carried out using the JEV-double antigen sandwich (DAS) ELISA. Oropharyngeal swabs were subjected to one-step RT-PCR to detect WNV RNA, in which positive reactions were subsequently sequenced. WNV seropositive rate of 18.71% (29/155) at 95% CI (0.131 to 0.260) and molecular prevalence of 15.2% (16/105) at 95% CI (0.092 to 0.239) were demonstrated in migratory and resident wild birds found in West Coast Malaysia. Phylogenetic analyses of the 16 WNV isolates found in this study revealed that the local strains have 99% similarity to the strains from South Africa and were clustered under lineage 2. Evidence of WNV infection in resident and migratory birds were demonstrated in this study. As a summary, intervention between migratory birds, resident birds and mosquitoes might cause the introduction and maintenance of WNV in Malaysia, however the assumption could be further proven by studying the infection dynamics in the mosquitoes present in the studied areas

Exposure to Zoonotic West Nile Virus in Long-Tailed Macaques and Bats in Peninsular Malaysia

The role of wildlife such as wild birds, macaques, and bats in the spreading and maintenance of deadly zoonotic pathogens in nature have been well documented in many parts of the world. One such pathogen is the mosquitoes borne virus, namely the West Nile Virus (WNV). Previous research has shown that 1:7 and 1:6 Malaysian wild birds are WNV antibody and RNA positive, respectively, and bats in North America may not be susceptible to the WNV infection. This study was conducted to determine the status of WNV in Malaysian macaques and bats found in mangrove forests and caves, respectively. Archive sera and oropharyngeal swabs from long-tailed macaques were subjected to the antibody detection using WNV competitive enzyme-linked immunosorbent assay (c-ELISA) and WNV RNA using RT-PCR, respectively, while the archive oropharyngeal and rectal swabs from bats were subjected to RT-PCR without serological analysis due to the unavailability of serum samples. The analysis revealed a WNV seropositivity of 29.63% (24/81) and none of the macaques were positive for WNV RNA. Meanwhile, 12.2% (5/41) of the bats from Pteropodidae, Emballonuridae, and Rhinolophidae families tested positive for WNV RNA. Here, we show a high WNV antibody prevalence in macaques and a moderate WNV RNA in various Malaysian bat species, suggesting that WNV circulates through Malaysian wild animals and Malaysian bat species may be susceptible to the WNV infectio

Sero-epidemiology of Japanese encephalitis virus among livestock, avian and companion animals in selected states of Malaysia

Japanese encephalitis (JE) is vector-borne disease causes encephalitis in human and horse as well as reproductive failure in sows. Pigs, bats, wild boar, and ardeidae birds play an important role as a main vertebrate amplifier of JEV. In Malaysia the disease is endemic in east Malaysia, Sarawak and epidemic in Peninsular Malaysia. The encephalitis cases were observed among human and animals reported in earlier 1950s. However, they are no data regarding seroprevalence pattern among livestock (buffaloes, pigs, cattle), avian (captivity water birds, poultry and migratory birds) and companion animals (cats, dogs). At the same time, risk factors associated with JEV seroprevalence in high risk areas in Malaysia are not documented. In this study, the objectives are to determine JEV seroprevalence rate, risk factors associated with JEV seroprevalence and determination of JEV genotype circulating among livestock (buffaloes, pigs, cattle), avian (water captivity birds, poultry and migratory birds) and companion animals (cats, dogs) were documented. Total of 461 serum samples were collected from dogs, cats, captivity water birds, village chickens, domestic pigs, buffaloes and cattle in selective states of Perak, Selangor and Sabah and screened for evidence of JEV antibodies by using a double sandwich IgG ELISA kits (DAS-ELISA). The risk factors associated with JEV seroprevalence of the animals were analysed by using IBM SPSS 22 which included Chi-square (χ2 test is not appropriate if one of expected cell value is less than 5, Fisher’s exact test was used at α = 0.05) and multiple logistic regression. A total of 13 survey criteria which include gender (male vs female), health status (healthy vs sick), age (young vs adult), breeding types (local, vs import), ownership of animal (own, shelter or none) and source of animals (same area, same district, same state vs epidemic areas) were collected. At the same time, environmental factors of sampling areas including presence of stagnant water (yes or no), paddy cultivation (yes or no), presence of ardeid birds (yes or no), locality (urban vs rural) and presence of mosquitos (yes or no). In the molecular assay, total of 791 samples of plasma, serum, and buffy coat from livestock (buffaloes, pigs, cattle), companion animals (cats, dogs) and avian (water captivity birds, poultry and migratory birds) were subjected to one step RT-PCR to detect the JEV by targeting the conserved region of NS3 of the JEV. In seroprevalence study among the animals studied, dogs show the highest seropositive rate of 80% (36/45; 95%CI: ±11.69) followed by pigs with 44.4% (40/90; 95% CI: ±1.715), buffaloes with 33.3% (15/45; 95% CI: ± 6.661), cattle with 32.2% (29/90; 95% CI: ±1.058), avian 28.9% (13/45; 95% CI: ±5.757), and cats with 14.4% (13/90; 95% CI: ±7.38). This study reveals that the risk factors associated with JEV seroprevalence are varied among different species of animals. The risk factors involved in different species of animals can be either singly or more than one. In additional to that, geographical factors may influence on the seropositivity of animals to JEV. In dogs, significant risk factor associated with JEV seroprevalence is the source of dog. In cats, significant risk factors associated with JEV seroprevalence are the age, locality, breeding types, and source of cats. In cattle, significant risk factors associated with JEV seroprevalence are gender, healthy status, breeding types and source of cattle. In pigs, significant risk factors associated with JEV seroprevalence are age and ownership of pigs. In buffaloes, significant risk factors associated with JEV seroprevalence are the source of buffalo and breeding types. In avian, significant risk factors associated with JEV seroprevalence are age and the breeding types. In this study, the high JEV seroprevalence in dogs observed is believed due to the preference and selective feeding pattern of Culex spp. in dogs compared to cats or human, thus making the dogs more prone to infection. JEV seroprevalence in pigs in this study are lower than reported previously probably due to the location of the subject matter in a non-paddy cultivation areas. This study also observed that JEV seroprevalence rate in buffaloes and cattle are low and support previous finding of this species play minimum role in JEV transmission cycles. The low viremia following JEV infection is too low of viral load to infect vector. Low JEV seroprevalence observed in chickens in this study is rather unexpected as avian was sampled in high risks areas with paddy plantation, water bodies and mosquitoes breeding sites. This finding may be further support by the lack of pig farming in this area, as the residents are engaged in fishing activities besides paddy cultivation. In this study, JEV antigen was unable to be detected in all the samples collected from various species of animals. Preliminary findings show that, all the animals were negative for JEV antigen. This could be due to the presence of antibodies of JEV naturalised the antigen. Apart from that, the sero-complex characteristics of flaviviruses make the antigen-antibodies cross-react with each other members. This makes the antigen being neutralised and unable to be detected in blood of the host. Samples collected during acute stage of JEV infection in animals have high chances of virus being isolated. In addition to that, the oronasal fluid is preeminent for isolation and detection of JEV antigen compared to serum, cerebrum spinal fluid, plasma and buffy coat